During the last year, we have established that in murine JLS-V9R cells, levels of both 2-5A-dependent RNase (subsequently referred to as the nuclease) and 2-5A-synthetase were closely linked to the cell growth rate; the enzymes increased during cell growth inhibition, and they decreased when cell growth was stimulated. Furthermore, the induction of the enzymes during confluency was not due to the spontaneous production of interferon. We could detect no interferon in the culture supernatants, and anti-interferon antibody did not prevent the induction of the nuclease during confluency. The induction of the enzymes preceded an inhibition of (3H)-thymidine incorporationby the cells into DNA by at least 24 hrs. These results are consistent with a role for the 2-5A-system (and, therefore, for the 2-5A-dependent RNase) in the inhibition of cell growth in JLS-V9R cells. We have shown that the 2-5A-dependent RNase gene(s) is regulated during differentiation of murine embryonal carcinoma cells. An induction of 2-5A-dependent RNase by interferon was demonstrated in each of three differentiated cell types by analyzing rRNA breakdown following the introduction of 2-5A into the intact cells. In contrast, in three undifferentiated cell lines there was no detectable 2-5A-dependent RNase either with or without interferon pretreatment. Because the undifferentiated cells are resistant to interferon action, these results support a role for the 2-5A-system in the antiviral and cell growth inhibitory activities of interferon. To clone cDNA for 2-5A-dependent RNase, polyA+ RNA was isolated from interferon-treated JLS-V9R cells and used to prepare a cDNA library in lambda-gt11. In addition, a sensitive and convenient screening procedure was developed for detecting clones producing the nuclease; the nuclease in phage plaques would bind [32P]-2-5A and result in a radioactive spot on a filter. These studies are providing fundamental information on the regulation of expression of 2-5A-dependent RNase during interferon treatment, cell growth inhibition, and cell differentiation. The cloning of cDNA for the nuclease will lead to basic information concerning the regulation of the nuclease gene(s) and will permit the introduction of the nuclease cDNA into cell lines deficient in the nuclease. (N)